Proteomics is an emerging technology for the study of protein function that has arisen in the post-genomics era. As the entire genomes of different organisms of the animal, plant, prokaryotic and viral kingdoms are being sequenced, thousands of genes are being identified, and these genes encode an abundance of distinct proteins. These proteins, the proteome of an organism, are being studied for identification of new drugs or commercially-important bio-active molecules.
Now the search for better therapeutics is being driven by several factors which need to be discovered: the identity and role of protein targets whose function may be pivotal for disease progression; the molecules that interact with these protein targets to attenuate their function and cause a therapeutic effect; the key biochemical pathways and the manner in which proteins interact with each other, which is critical for the selection of the most appropriate target for therapeutic intervention; and the impact of therapeutic candidates on the whole organism, including the most common variants of important proteins to identify potential side-effects and toxicity (the pharmacogenomic profile) of drug candidates as rapidly as possible.
The traditional approaches to drug discovery are either by selective drug design methods, which to date are at best, an inexact science, or by screening test compounds in a disease model. Screening, historically, has been the most effective of the two methods for identifying therapeutics, but it is labor-intensive. Most screening has been of randomly-selected candidates. More recently, screening has included the use of combinatorial libraries of candidate drugs, or portion thereof. Generally however, the techniques used to screen libraries, such as liquid chromatography, have been slow and expensive.
Therefore, a need exists for an apparatus and method to screen candidate therapeutic drugs that significantly reduce of eliminate the above-mentioned problems.
The present invention is generally directed to a method for screening a proteome, which is a mixture of proteins or compounds derived from a common source, and an apparatus for screening a proteome.
In one embodiment, the method for screening a proteome includes passing the proteome though an array of elements that possess an affinity for at least one compound in the proteome. This is followed by washing the array to remove those compounds of the proteome that do not specifically bind, and subsequently directing an eluent through the array by centrifugal force, which releases the specifically-bound compound, and elutes the released compound, thereby screening the proteome.
In another embodiment, an apparatus is provided to screen a proteome. The apparatus includes an array of affinity elements that includes at least one ligand, and means to apply a centrifugal force to the array such that, in the presence of an eluent, the compound of the proteome that is bound to the ligand can be eluted from the affinity element by centrifugation.
This invention has many advantages. For example, proteomes can be screened rapidly and systematically, whereby not only new lead compounds can be identified, but also the identity of the physiological targets of known drugs. Use of an affinity array enables efficient and quantitative capture of proteins, even when proteins are present in small quantities. This is important since disease-causing proteins, such as transcription factors or kinases, are often present in low copy numbers in a cell. Furthermore, the amount of protein captured can be directly measured. The use of small bed volumes of resin enables the enrichment of targeted proteins and proteomes. By employing centrifugal force to direct an eluent through the affinity array, the total volume of the eluent applied to the array can be recovered, which contrasts with flow-type elution methods that generally result in larger elution volumes and therefore greater dilution of the eluate. Thus, relatively small volumes of eluents can be applied to the affinity array, which can result in significantly more efficient recovery of proteins that are readily amenable to subsequent manipulation. For example, because the eluted proteins are concentrated in a small volume, they typically can be directly sampled for MALDI-TOF mass spectrometry or ICAT analysis without further manipulation. This generally provides for more rapid identification of targets. Furthermore, potential toxicity can be assessed by sequencing proteins that are simultaneously eluted. Unlike many known methods of designing improved therapeutic compounds, this invention can also be used to identify physiological targets of known drugs, the mechanism of action of which was previously unknown. By identifying the physiological target of the known drug using this invention, the mechanism of action can be assessed and improved targets can be identified. Furthermore, this invention can be automated and integrated into a robotics system.